In vitro susceptibility testing of dermatophytes with their fragmented mycelia as inoculum


  • V. A. Czaika Universitats medizin Berlin, Clinics for Dermatology, Venereology und Allergology, Charite, Campus Benjamin
  • A.F. Schmalreck Universitatsmedizin Berlin, Clinics for Dermatology, Venereology und Allergology, Charite, Campus Benjamin



dermatophytes, susceptibility testing, inoculum, fragmented mycelium, azoles, echinocandins, topical antifungal agents


377 clinical isolates and seven dermatophyte culture collection strains were tested in vitro by microdilution against a panel of 18 antifungal agents. A newly introduced method for rapid and reliable inoculum preparation for moulds using the fragmented mycelia of freshly grown isolates as inoculum was firstly applied to dermatophytes. The performance of the method was tested with two different culture media which are recommended by different standardized testing methods for moulds. The standardized fragmented mycelia provided both, countable single colonies (viable units) on solid culture media and distinct readable endpoints (minimum inhibitory concentrations) in the microdilution wells for all tested topical and systemic drugs, including the echinocandins. As with moulds, and as shown by the culture collection strains, reproducible minimum inhibitory concentrations were obtained with an essential agreement (± 1 log2-dilution) of 97% to 100%, by a significant reduction of the overall testing time. As exemplarily shown for the microdilution technique, this inoculum method should also provide a solid basis for improved agar-based susceptibility testing methods such as disc-, tablet-, or strip-tests.


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Emmons, C.W. 1945. Fungicidal and fungistatic agents: proposed method for testing fungicides against Trichophyton. Am. Public Health, 35, 844-846.

Bergmann, S. 1955. In vitro studies on antimycotics. A comparison of different methods. Acta Pathol. Scand. Suppl. 104.

Roth, F.J., Sallman, B., and Blank, H. 1959. In vitro studies of the antifungal antibiotic griseofulvin. J. Invest. Dermatol. 33, 403-418.

Granade, T.C., and Artis, W.M. 1980. Antimycotic Susceptibility Testing of Dermatophytes in Microcultures with a Standardized Fragmented Mycelial Inoculum. Antimicrob. Ag. Chemother. 17, 725-729.

Aytoun, R.S.C., Campbell, A.H., Napier EJ, Seiler, D.A.L. 1960. Mycological aspects of action of griseofulvin against dermatophytes. AMA Arch. Dermatol. 81, 52-58.

Qualman, S.J., Jones, H.E., and Artis, W.M. 1976. An automated radiometric microassay of fungal growth: quantitation of growth of T. mentagrophytes. Sabouraudia, 14, 287-297.

Jessup, C.J., Warner, J., Isham, N., Hasan, I., and Ghannoum, M.A. 2000. Antifungal Susceptibility Testing of Dermatophytes: Establishing a Medium for Inducing Conidial Growth and Evaluation of susceptibility of Clinical Isolates. J. Clin. Microbiol. 38, 341-344.

Fernndez-Torres, B., Caba±es, F.J., Carrillo-Mu±oz, A.J., Esteban, A., Inza, I., Abarca, L., and Guarro, J. 2002. Collaborative evaluation of optimal antifungal susceptibility testing conditions for dermatophytes. J. Clin. Microbiol. 40, 3999-4003.

Ghannoum, M.A., Chatuverdi, V., Espinel-Ingroff, A., Pfaller, M.A., Rinaldi, M.G., Lee-Yang, W., and Warnock, D.W. 2004. Intra- and Interlaboratory Study of a Method for Testing the Antifungal Susceptibilities of Dermatophytes. J. Clin. Microbiol. 42, 2977-2979.

Young, C.N. 1972. Sensitivity patterns to griseofulvin of Trichophyton rubrum and other ringworm fungi. Trans. St. Johns Hosp. 58, 226-234.

Sekhon, A.S., and Funk, A. 1977. In vitro inhibitory studies of phacidin against some dermatophytes, opportunistic and systemic fungi, and yeasts pathogenic on man. J. Antimicrob. Chemother. 3, 95-100.

Favre, B., Hofbauer, B., Hildering, K.-S., and Ryder, N.S. 2003. Comparison of In Vitro Activities of 17 Antifungal Drugs Against a Panel of 20 Dermatophytes by Using a Microdilution Assay. J. Clin. Microbiol. 41, 4817-4819.

Ciebiada-Adamiec, A., Ciebiada, I., Malafiej, E., and Denys, A. 2008. Susceptibility testing of dermatophytes - proposition of inoculum standardization. Mikologia Lekarska, 15, 17-19.

Shathele, M.S., and Fadlelmula, A.. 2010. In vitro Effectivness of Some Antifungal Drugs in Treatment of Trichophyton verrucosum; Dermatophytic Fungi. Asian J. Anim. Vet. Adv. 2010, 1-13.

Korting, H.C., Ollert, M., Abeck D. 1995. Results of a German Multicenter Study of Antimicrobial Susceptibilities of Trichophyton rubrum and Trichophyton mentagrophytes Strains Causing Tinea Unguium. Antimicrob. Ag. Chemother. 39, 1206-1208.

CLSI M38-A2. 2008. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard Second Edition. Clinical Laboratory Standards Institute, USA.

EUCAST Definitive Document E.DEF 9.1. 2012. Method for determination of broth dilution minimum inhibitory concentration of antifungal agents for conidia forming moulds.

Schmalreck, A., Willinger, B., Czaika, V., Fegeler, W., Becker, K., Blum, G., and Lass-Fl, C. 2012. Susceptibility Screening of Hyphae-Forming Fungi with a New, Easy, and Fast Inoculum preparing Method. Mycopatholgia 174, No. 1, July 2012: DOI 10.1007/s11046-012-9570-7.

Schmalreck, A.F., Fegeler W., Becker K., Lass-Fl C., and Czaika, V. 2014. Cross-Resistance of Azoles, Echinocandins, Flucytosine, and Amphotericin B in Clinically Important Hyphomycetes. International Journal of Microbiology Research, ISSN: 0975-5276 & E-ISSN:0975-9174.

Bastert, J., Korting, H.C., Trnkle, P., and Schmalreck, A.F. 1999. Identification of dermatophytes by Fourier-Transform Infrared-Spectroscopy (FT-IR). Mycoses, 4, 525-528.

Schmalreck, A.F., Willinger, B., Haase, G., Blum, G., Lass-Frl, C., Fegeler, W., Becker, K., and the Antifungal Susceptibility Testing (AFST) Study Group. 2012. Species and susceptibility distribution of 1062 clinical yeast isolates to azoles, echinocandins, flucytosine and amphotericin B from a multi-centre study. Mycoses, 55, e124e137. DOI: 10.1111/j.1439-0507.2011.02165.x.

Arendrup, M.C., Garcia-Effron, G., Lass-F C., Gomez Lopez ,A., Rodriguez-Tudela, J.-L., Cuenca-Estrella, M., and Perlin D.S. 2009. Susceptibility testing of Candida species to echinocandins: comparison of EUCAST EDef 7.1, CLSI M27A3, Etest, disk diffusion and agar-dilution using RPMI and IsoSensitest medium. Antimicrob. Agents Chemother. 54, 426439.

Guidry, D.J., and Trelles, G.H. 1962. Evaluation of a new Method for the Preparation of Homogenous Mycelial Suspensions. J. Bacteriol. 83, 53-60.

Savage, G.M., and Vander Brook, M.J. 1946. The frangmentation of the Mycelium of Penicillium notatum and Penicillium chrysogenum by a HighSpeed Blender and the Evaluation of Blended Seed. J. Bacteriol. 52, 385-391. [25] Burden, D.W. 2008. Guide to the Homogenization of Biological Samples. Random Primers 7, 1-14.

Schmalreck, A.F., and J. Hoi 1984. Offenlegungsschrift DE 3308325 A1: Verfahren und Vorrichtung zur vollstndigen oder partiellen Zersrung von Mikroorganismen. Deutsches Patentamt M¼nchen.

Zhang, J., Chen, J., Huan, H.-G., Xi, L.-Y., Lai, W., Xue, R.-Z., Zhang, X.-H., and Chen ,R.-Z. 2010. Comparison of a glucose consumption based method with the CLSI M38-A method for testing antifungal susceptibility of Trichophyton rubrum and Trichophyton mentagrophytes. Chin. Med. J. 123, 1909-1914.

Barchiesi, F., Arzeni, D., Camiletti, V., Simonetti, O., Cellini, A., Offidani, A.M., and Scalise, G. 2001. In Vitro Activity of Posaconazole against Clinical Isolates of Dermatophytes. J. Clin. Microbiol. 39, 4208-4209.

Bejak, V. 1985. Standarization of Hyphal Inoculum of Aspergilli for Amphotericin B Testing. J. Clin. Microbiol. 21, 509-512.

Nweze, E.I., Mukherjee, P.K., and Ghannoum, M.A. 2010. Agar-based Disk Diffusion Assay for Susceptibility Testing of Dermatophytes. J. Clin. Microbiol. 48, 3750-3752.

Santos, D.A., Barros, M.E.S, and Hamdan, J.S. 2006. Establishing a method of inoculum preparation for susceptibility testing of Trichophyton rubrum and Trichophyton mentagrophytes. J. Clin. Microbiol. 44, 98-101. [32] Santos, D.A., and Hamdan, J.S. 2005. Evaluation of Broth Microdilution Antifungal Susceptibility Testing Conditions for Trichophyton rubrum. J. Clin. Microbiol. 43, 1917-1920.

Fortwendel, J.R., Juvvadi, P.R., Pinchai, N., Perfect, B.Z., Alspaugh, J.A., Perfect, J.R., and Steinbach, W.J. 2009. Differential effects of inhibiting chitin and 1,3-{beta}-D-glucan synthesis in ras and calcineurin mutants of Aspergillus fumigatus. Antimicrob. Agents Chemother. 53: 476-482. doi: 10.1128/AAC.01154-08.

Espinel-Ingroff, A., Canton, E., and Penman, J. 2009. Updates in antifungal susceptibility testing of filamentous fungi. Cur Fungal Infect Rep. 3, 133-41.

Bao, Y.Q., Wan, Z., and Li, R.Y. 2013. In vitro antifungal activity of micafungin and caspofungin against dermatophytes isolated from China. Mycopathologia, 175, 141-145. doi: 10.1007/s11046-012-9571-6.

Pound, M.W., Townsend, M.L., Richard, H., and Drew, R.H. 2010. Echinocandin pharmacodynamics: review and clinical implications. J. Antimicrob. Chemother. 65, 1108-1118. doi: 10.1093/jac/dkq081.

Gomez-Lopez ,A., deOliveira, A.N., de Oliveira, L.A., and Andrade, J.S. 2010. Partial characterization of amylases of two indigenous Central Amazonian rhizobia strains. Braz. Arch. Biol. Technol. 53, no.1 Curitiba Jan./Feb. 2010.

Fernndez-Torres, B., Carrillo, A.J., Martn, E., Del Palacio, A., Moore, M.K., Valverde, A., Serrano, M., and Guarro, J. 2001. In vitro activities of 10 antifungal drugs against 508 dermatophyte strains. Antimicrob. Ag. Chemother. 45, 25242528.

Siqueira, E.R., Ferreira, J.C., dos Santos, P.R., Lavrador, M.A.S., and Candido, R.C. 2008. Dermatophyte Susceptibilities to Antifungal Azole Agents Tested by In Vitro Broth Macro and Microdilution Methods. Rev. Inst. Med. Trop. S. Paulo, 50, 1-5.

Jha, B.K., Murthy, S.M., Mathur, M., and Gupta,. B.P. 2013. Antifungal Susceptibility Testing of Dermatophytes from all Tinea cases by Broth Micro Dilution Method. J. Pharm. Res., 2, 1-4.

Arajo, C.R., Miranda, K.C., Fernandes, O. deFatima, L., Soares, A.J., Silva, M., and do Rosario, R. 2009. In vitro susceptibility testing of dermatophyte isolates in Goiania, Brazil, against five antifungal agents by broth microdilution method. Rev. Inst. Med. Trop. S. Paulo, 51, 1-12.

Pujol, I., Capilla, J., Fernandez-Torres, B., Ortoneda, M. And Guarro, J. 2002. Use of the sensititre colorimetric microdilution panel for antifungal susceptibility testing of dermatophytes. J. Clin. Microbiol.,40, 2618-2621.



How to Cite

Czaika, V. A., & Schmalreck, A. (2013). In vitro susceptibility testing of dermatophytes with their fragmented mycelia as inoculum. JOURNAL OF ADVANCES IN BIOLOGY, 6(1), 775-788.